Methods of controlling mycotoxin production using certain dialkyl enol phosphates

ABSTRACT

MYCOTOXIN PRODUCTION BY A FUNGUS IS CONTROLLED OR INHIBITED BY SUBJECTING THE FUNGUS TO AN EFFECTIVE AMOUNT OF A DIALKYL ENOL PHOSPHATE.

Unit (I St P O a Patented Mar. 19, 1974 TABLE-Continued 3,798,323 S M tMETHODS OF CONTROLLING MYCOTOXIN PRO- Pwes' DUCTION USING CERTAINDIALKYL ENOL Aspergillus flavus Aflatoxln B afiatoxln G PHOSPHATESafiatoxin B aflatoxin 6;; John S. Leary, In, Alamo, Califi, assignor toShell Oil aflatoxin M aflatoxin Company, Houston, Tex. M aspergillicacid; as- No Drawing. Filig Agggsi 1997/1,6Ser. No. 174,975 pertoxin.

0 n 3 Rubratoxin B. 424-212 11 Claims Penicillium rubrum Cyclopiazonicacid.

Penicillium cyclOpium Ochratoxin A.

ABSTRACT OF THE DISCLOSURE Mycotoxin production by a fungus iscontrolled or inhibited by subjecting the fungus to an effective amountof a dialkyl enol phosphate.

BACKGROUND OF THE INVENTION This invention relates to a method ofcontrolling or inhibiting mycotoxin production by fungi. Moreparticularly, this invention relates to a method of controlling orinhibiting the production of mycotoxins by fungi of the class FungiImperfecti by subjecting the fungi to an effective dosage of a dialkylenol phosphate.

Thoughout the world each year, a considerable portion of the worlds foodsupply is destroyed by the action of various fungi and bacteria. Inaddition, fungi produce various substances refered to as mycotoxins,which are poisonous to warm blooded animals. Mycotoxins are produced onfoodstuffs under certain conditions of temperature, oxygen level,nutrient type and moisture content and are of particular concern to thefood industry, especially those concerned with the production of cerealgrains and nuts.

Fungi, such as those of the genus Aspergillus, themselves are not toxicand even the species Aspergillus flavus in the soil will not usuallyinvade the growing plant or its seeds until the shell, husk or epidermisof the plant or seeds have become damaged. However, when the invasiondoes take place in a few seeds, enough mycotoxins can be produced tomake a large store of seeds toxic to warm blooded animals. The nature ofthis protective mechanism in the shells and husk of the nuts or seeds isnot known. It is known, however, that once this protective coating ormembrane has been damaged that the seed becomes subject to the fungiwhich will then produce mycotoxins.

Typical mycotoxins produced by the class Fungi Imperfecti and theprincipal producing species are listed in the following table.

TABLE Species: Mycotoxin Fusarium tricinetum- Sporofusariogenin.Cladosporium epiphylum Epi-cladosporic acid. Cladosporium: fagiFagicladosporic acid.

Penicillium islandz'cum Islanditoxin; luteoskyrin. Penicillium rugulosumRuguiosin.

Penicillium tardum Ruguiosin.

Penicillium brenneum Ruguiosin.

Penicillium urticae Patulin.

Penicillium citrinum Citrinin.

Penicillium notatum Xanthocillin.

Penicillium citreoviride Citreoviridin.

Aspergillus fumigatus Fumagillin; gliotoxin; helvolic acid.

Aspergillus terreus Terreic acid.

F usarium' graminearum Zearalenone.

Aspergillus ochraceus Trichodermin.

toxin; 4 acetamido-4- hydroxy 2 butenoic Fusarz'um roseum var.acid-gamma-lactone.

graminearium F-2 toxin.

Fusarium tricinctum The mycotoxins of particular concern throughout theworld at present are the afiatoxins. Aflatoxins are primarily the toxicmetabolites produced by the class Aspergillaceae and particularly by thegenera Aspergillus and Penicillium. The primary producer of aflatoxinsis the species Aspergillus flvus (as noted in the table above) marilythe toxic metabolites produced by the class .Aspergillaceae andparticularly by the genera Aspergillus and of mycotoxins known and evenin low dosages are potent heptaocarcinogens. In addition, aflatoxinshave been demonstrated in test animals to reduce fertility, producemalformations in offspring and cause liver damage.

Aflatoxins are designated B B G G M and M Aflatoxins B B G and G areproduced by the fungi and are named on the basis of the color offluorescence on thin layer chromatography plates. The letter B thusstands for blue fluorescence and the letter G stands for greenfluorescence. The M aflatoxins are animal metabolites'of the B and Gaflatoxins and are referred to as milk toxins.

These aflatoxins possess the following chemical structures:

I u @152 to 3 7 DESCRIPTION OF THE INVENTION It has now been found thatmycotoxin production can be controlled or greatly inhibited bysubjecting the mycotoxin producing fungi to an elfective dosage of adialkyl enol phosphate of the formula:

wherein R and R are C -C alkyl and may be the same or diiferent, A is Hor CH Y is H or halogen and Z is halogen or By halogen is meant middlehalogen and particularly chlorine or bromine with chlorine beingpreferred.

Preferred are those compounds of the formula:

wherein R and R are as defined above and R" is hydrogen or chlorine.Especially preferred is dimethyl 2,2-dichlorovinyl phosphate hereinafterreferred to as DDVP, and diethyl 2-chlorovinyl phosphate.

The mechanism by which this invention operates is not one of fungicidalactivity in that the growth of fungi is not prevented. Highconcentrations of the dialkyl enol phosphates do not appear to inhibitthe growth of the fungi but have been observed to prevent or suppresssporulation. Studies conducted using lower concentrations of the enolphosphate indicate that neither mycelia growth nor sporulation wereaffected; however, the amount of aflatoxin produced was greatlyinhibited and in some cases no measurable mycotoxin was produced at all.

These enol phosphates may be administered in a variety of ways accordingto the substrate that is to be protected from mycotoxin formation. Theenol phosphate can be applied directly to the fungi or to the substrateto be protected. Moreover, the enol phosphate may be administered as aspray, vapor, dust, solution, etc. For example, when applying directlyto the fungi, the enol phosphate may be poured or sprayed or otherwiseplaced upon the fungi as a solution. When cereal grains or nuts and thelike are to be protected against mycotoxin production by invading fungi,the grain or nuts or whatever the substrate is, may be treated byapplying the enol phosphate as a vapor or the substrate can be submergedin a dilute solution of the enol phosphate and then removed and dried.

While this invention is not limited to any particular method ofapplication, it will be obvious to those skilled in the art that theeasiest method of application is to apply the enol phosphate in the formof a vapor. For example, the enol phosphate may be made up as an aerosolunder pressure, which when the pressure is released, releases activeingredient into the atmosphere, or the vapor may be generated by meansof the application of heat to the appropriate enol phosphate in order tovaporize it. The most preferred method of application is to us aresinous generator such as those described in US. Pat. 3,318,769. Inthat application, enol phosphates are incorporated into resinouscompositions such as those comprising a plasticized polyvinyl chloride.

The concentration of dialkyl enol phosphates to be used is dependententirely upon the method of application and upon the substrate to beprotected. For example, it is obvious that a 1,000 cu. ft. roomone-third full of a substrate such as peanuts or cofiee beans wouldrequire less of the active ingredient than would the same room twothirdsfull. In general, concentrations of 1 to 20 ppm. of active ingredient onthe substrate will be effective. However, depending upon the degree ofcontrol desired, higher concentrations may be employed.

The invention will be described more particularly with reference to thefollowing examples.

EXAMPLE I (A) Afiatoxin production and extraction Aspergillus flavusvar. parasiticus, ATCC NO. 15517, known aflatoxin producer, was employedand stock cultivated on Difco Sabouraud maltose agar slants grown at 25C. 25 grams of shredded wheat were placed in wide-mouth, glass,screw-top jars and sterilized, after which 50 milliliters of sterilewater was added to each. One milliliter of spore suspension from a stockslant was inoculated onto this substrate and incubated at 27 C., 70%relative humidity, with the vessel covers very loosely attached.

Two separate tests were conducted. In one test 10.0 grams of a resingenerator strip comprising 70% polyvinyl chloride (PVC) 1()% dioctyladipate (DOA), and 20% dimethyl 2,2-dichlorovinyl phosphate (DDVP), weresuspended above the inoculated substrate and the covers to the jarsloosely attached. In this test control jars containing PVC resinpl-asticized by DOA but containing no DDVP were incubated in separatechambers under identical conditions. In the other test, open jars wereincubated in a 512 cubic foot environmental chamber at 27, relativehumidity, which had been pre-equilibrated with a DDVP-PVC resin strip.

Concentration of DDVP in air samples were determined by standardmethods. 20 liter volumes were routinely sampled. The DDVP concentrationin terms of millig rams per liter was determined for the glass jars byflushing a three-neck distilling flask with 20 liters of air which wasincubated under identical conditions to the wide-mouth jars. The DDVPconcentration was found to be 216 micrograms per liter. In the 512 cubicfoot room equilibration was brought about by introducing the DDVP-PVCstrips into the chamber three days prior to the beginning of incubationand the average concentration of DDVP in the air within the chambervaried from an average of 3.53 micrograms per liter at the beginning ofincubation, to 2.05 micrograms per litre on the seventh day ofincubation.

Following incubation, the cultures were extracted for afiatoxin byadding milliliters of chloroform at 55- 60 C. and shaking on areciprocating shaker for twenty minutes at room temperature. Thechloroform extract was poured through cheesecloth and filtered throughglass wool and Whatman No. 1 filter paper. This procedure was repeatedthree times, and the filter paper and glass wool were then rinsed with aminimal amount of acetone. The filtrate was pooled, measured andanalyzed for aflatoxin.

(B) Fluorometric analysis for afiatoxin Aflatoxin was quantitated fromstandards of aflatoxins B and G in chloroform, applied to thin layerchromatography (TLC) plates and developed in solvent of 97:3 CHCI :CH OH(parts per volume). TLC plates were scanned automatically in a TurnerModel IH fiuorometer.

Table I depicts the influence of DDVP on the production of aflatoxin asdetermined fluorometrically.

TABLE I [The influence of DDVP on the production of afiatoxln byuspergillus flmma B1 Gr B1 and G1 DDVP concen- X fold X fold X foldtration 5 Mg. Percent decrease Mg Percent decrease Mg. Percent decreaseControl 0. 17. 100 7. 5 100 25 100 512 cu. ft. hut 3. 5-2. 0 1.0 5.717.5 0.25 3. 33 3O 1. 25 5 20 Glass jars 216 0. 3 1. 7 58. 3 0. 20 2. 6737. 5 0. 5 2 50 l Determined at the end of six days incubation.Mierograms/liter;

As is evident from the above table, the lower concentrations of DDVP,i.e., 2 to 3 /1 micrograms per liter air space, resulted in a decreaseof the B and G afiatoxin some 17.5-30 times, whereas at the higherconcentrations in the covered glass jars, the decrease was significantlygreater, i.e. 58.3 and 37.5 times.

(C) Biochemical analysis for aflatoxin To corroborate the above effects,as determined by analytical chemical methods, a bioassay using chickenembryo mortality in embryonating eggs was performed. The filtrate thatwas pooled and measured for fluormetric analysis of aflatoxin wastreated by evaporating the chloroform under free-flowing air. Theresidue was suspended in 15 milliliters of propylene glycol andsterilized by passage through a 0.45 micron filter prior to biologicalassay. The biological assay method used was similar to that adopted byVerrett et al. in J.A.O.A.C. 47, 1003-1006 (1964). The bioassays werebased upon the mortality resulting from injection of the cultureextracts into fertile White Leghorn chicken eggs via the air sac route.Egg inoculation was performed by drilling through the shell at the airsack, injecting 0.05 milliliter of the sterilized propylene glycolextract and sealing the shell with molten wax. The eggs were thenincubated within an incubator at 101 F. and candled at 5 days and daysto determine the number of viable embryos Groups of 30 eggs were usedper dose level sample and mortality data were corrected from the numberof infertile eggs The inoculations were made from the extracts obtainedby extracting the aflatoxin from the glass jars containing 10 grams PVC-DDVP resin strips. The inoculations were made using the undilutedextract and extracts diluted with propylene glycol at dilutions of 1-20,1-40, 1-80, 1-60 and 1-240. The results are reported in Table H.

From the above results it is evident that the positive controlcontaining no DDVP caused chick embryo mortality even at a dilution of 1part to 240, whereas the group containing the DDVP produced no mortalityat dilutions of 1 to 240. This data corresponds with the data containedin Table I which shows that there is considerably less aflatoxinproduced in the presence of DDVP, and hence lower mortality atcorresponding dilutions of the extract when inoculated into chickeneggs.

A further observation made from the above tests was that at theconcentration of 216 micrograms per liter of DDVP, the DDVP did notinhibit the mycelial growth but did prevent spore formation in theAspergillus flavus. However, at the lower air concentrations, i.e.,3.5-2.0 micrograms per liter there was no visible inhibition of mycelialgrowth or sporulation although a marked decrease in aflatoxin productionwas still observed.

EXAMPLE II The procedure used in Example I was followed for producingaflatoxin, extracting it, and inoculating eggs to determine the chickenembryo mortality. The results are recorded in Table III.

TABLE III Embryo mortality Test group 5th day 10th day A. flaws positivecontrol 20/20 A. flaws plus blank PVC strip 14/15 14/15 A. flaws plusDDVP-PVC strip 0/17 0/17 Number of eggs killed over number of fertileeggs on test.

This table confirms the results obtained in Example I. During this testsporulation was abundant in the positive control culture and in theblank strips containing no DDVP. However, no evidence of sporulationcould be detected on a mycelial mat which developed in the DDVPatmosphere.

EXAMPLE HI Another test following the procedures of Example I wascompleted, with the results being reported in Table IV.

These results again confirm the results contained in Examples I and IIand show the marked activity on the part of DDVP by significantlyreducing aflatoxin production, spore prevention was again evident inthis test.

EXAMPLE IV Again following the procedure of Example I, an additionalstudy was made to confirm the results of the preceding tests. Inaddition to inoculating the chicken eggs with undiluted extract,dilutions of 1/5, 1/10 and 1/20 were also made. The results of thisstudy are reported in Table V.

a 10 gram strips.

EXAMPLE V Fusarium rosewm var. graminearum, known producer of mycotoxinF-2, was cultured on a mixture of wheat and rice. The positive controlsample contained no DDVP whereas the sample under test contained partsper million by weight of DDVP. Both samples were incubated for six weeksat 28 C. and for eight Weeks at 10 C. At the conclusion of theincubation period the samples were extracted by standard procedures andtested for F2 concentration. The results are recorded in Table VI.

TABLE VI F-Z concentration (op- From this example it is evident that theenol phosphates, DDVP in particular, are etfective in inhibiting theproduction of mycotoxins other than afiatoxins.

In the following three examples the fungus Aspergillus flavws NRRL 2999was cultured on corn, wheat, rice and/ or shelled peanuts. The cerealgrains had a moisture content of about 20% whereas the moisture contentof the peanuts was about 17.3%. All of these foodstufi's were autoclavedfor 30 minutes at 120 C. and shaken to prevent caking and thenreautoclaved 24 hours later.

DDVP was diluted with water so that the desired residue deposits wouldresult when 1 milliliter of the dilution was applied per sample. UniformDDVP applications were achieved by tumbling the treated sample forseveral minutes in a gallon jar. Both treated and control samples weretransferred in 100 gram lots to separate sterile flasks, wherein eachwas inoculated with 0.1 gram of a presoaked soil culture of Aspergillusflavus and incubated 7 days at about 27 C. and 70% relative humidity.Quantitative determinations of aflatoxin were then made.

EXAMPLE VI In this example the foodstufis were first treated with DDVPat 20 parts per million by weight and were then inoculated with theAspergillus flavus with the results being reported in Table VII.

TABLE VII .Aflatoxin concentration (p.p.m.) in- Presence confirmed bythin-layer chromatography but not measurable by UV spectrophotometry.

8 These results show that DDVP residues of 20 parts per million byweight are highly eifective against the production of aflatoxin onwheat, corn and peanuts and significantly reduced aflatoxin productionon rice.

EXAMPLE VII In this example wheat was treated so as to have DDVPresidues of 0, 5, 10 and 20 parts per million by weight on separatesamples which were then inoculated with Aspergillus flavus. The resultsare recorded in Table VIH.

Presence confirmed by thinlayer chromato raph but not measurable by UVspectrophotometry. g y

The data presented in this example shows that even at levels of 5 and 10parts per million by weight DDVP significantly reduces the aflatoxincontent over corresponding untreated controls.

EXAMPLE VIlI This example shows the results of first inoculating wheatwith Aspergillus flavus and then 2 and 4 days later treating theinoculated samples with 20 parts per million by weight of DDVP.

TABLE IX Atlatoxin (p.p.m.) in wheat DDVP treatment followinginnoculation after 7 days incubation Days Amount added (p.p.m.) Run 1Run 2 Run 3 l N o aflatoxin in untreated control when DDVP was applied.b Aflatoxm in untreated control avg. 29.3 parts per million by weightwhen DDVP was applied.

From the above it is evident that the application of DDVP residuesreduced aflatoxin production on wheat even when applied 2 or 4 daysfollowing inoculation of the wheat with the fungus.

EXAMPLE IX This example shows typical enol phosphates that may be usedto inhibit or prevent the formation of aflatoxins. The compounds to betested were suspended into 50 milliliters of sterile water and added tojars containing 25 grams of sterilized shredded wheat to give a finalconcentration of 10 parts per million by weight of compound in theshredded wheat substrate. The jars were inoculated with A. flavus,capped and placed in an enclosure maintained at F. for 6 days. Theaflatoxin was extracted with chloroform and analyzed by standardtechniques with the following results.

TABLE X CzHsO O O CHaO O \{1 ll Aflatoxin content CH=CHCO CH; P 0 0:0 Ch

Compound (mieigggramslmlg l CfH O C4H 0 I claim as my 1nvent10n:irilllfitlilil it fifififfiffffij:::::::::::::::::::::: A math 0finhibiting mYCOtOXiH Production of DDVP,inflPI1latPfl fungi of the classFungi Imperfecti which comprises ap- CHaO O plying to the fungus of thatclass a dosage effective to \II control the production of mycotoxin of acompound POOH=CHC1 inoculated of the formula:

01130 R C2H5 \fi o 0 Y roon=cc1= inoculated l 011150 5 A CH3O\(") tPOC=CHCOCH= mullet where R and R each is C -C alkyl, A is hydrogen orCHaO 0H, methyl, Y is hydrogen or chlorine and Z is chlorine or I Toolow to determine. F;

0 EXAMPLE X 2. The method according to claim 1 wherein A is Following aprocedure similar to that disclosed in Exhydrogen and Z 15ehlorirleample 1 except suspending h compound in 110 ini- 3. The methodaccord ng to claim 2 wherein R and liters of sterile water and using achemical solution coneach 18 methyl and 1S q taining glucose instead ofshredded wheat as substrate, the The Flethed aeeofdlng t0 elalm 3wherein the fungus following results were obtained. Each test wasallowed to 15 Aspergllllls fl run for 7 days with one set of containersbeing agitated e method according to elalm 3Whefe1l1the fungus while aduplicate set was allowed to remain standing still. Fusarlum m-Yeumflmmmearllfrl- The samples were then analyzed for aflatoxin andcommethod Of 0131111 1 Yvhel'eln the compound 15 pared to an inoculatedcontrol. The results are reported pp t0 the g s as a 801M101!- in TableXI. 7. The method according to claim 1 wherein the com- TABLE XI poundis applied to the fungus in the vapor phase.

8. The method according to claim 7 wherein the comfgfi figfi pound isincorporated in a plasticized polyvinyl chloride resin. Compound 38%? ggg Standing 9. The method according to claim 1 wherein R and R each ismethyl, A is methyl, Y is hydrogen and Z is DDVP 10 100 100 40 o \Hmethyl.

PCH=GHC1 10. The method according to claim 2 wherein R and o R' each isethyl and Y is hydrogen. 11. The method according to claim 1 wherein thefungus is present on a substrate. CHaQ 0 0 10 7o i 0C=CH( i0CHiReferences Cited onto on; UNITED STATES PATENTS DDVP-PVC pellets 20 100so 3,130,120 4/1964 Schultz et a1 424-219 3,071,610 1/1963 Senkbeil424219 The following compounds may also be used to inhibit $318,7695/1967 Folckemer et 424 83 X or prevent mycotoxin production.

CHaO O aHrO ALBERT T. MEYERS, Primary Examiner L. SCHENKMAN, AssistantExaminer US. Cl. X.R. 424-2 19 P -w UNITED STATES PATENT OFFICE (5/69)CERTIFICATE OF CORRECTION Patent No. 3,798,323 Dated March 19, 1974Inventofls) John g. Learyl Jr.

It is certified that errcr appeara in the above-identified patent andthat said Letters Patent are hereby corrected as shown below:

In claim 1, that part of the claim appearing at lines 20-22, reading"-80" shQuld read -E-o methyl.

In claim 9, that part of the claim appearing O O at lines 40-42, reading"-210" should read O methyl.

cancel "methyl." at line 43.

Signed and sealed this 28th day of January 1975.

(SEAL) Attest:

M. GIBSON JR. C. MARSHALL DANN fig zting Officer Commissioner of Patents

